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ObjectiveTo establish a method of two-times second derivative Fourier transform infrared spectroscopy （FTIR） for identifying melamine （MEL） and its conjugates with cyanuric acid （MEL-CYA） or with uric acid （MEL-UA） in human urinary calculi.MethodsMEL， MEL-CYA and MEL-UA were added to calcium oxalate stone samples， and then analyzed by two-times second derivative FTIR for identifying the characteristic response bands of MEL and its conjugates in stones as well as confirming the ratio of detection.ResultsThe second derivative FTIR could improve the detection of MEL in CaOx stones by two orders of magnitude. When CaOx being mixed with MEL （MEL∶ CaOx）， the two-times second derivative FTIR showed the characteristic peak at 1 548 cm-1 and the minimum mass ratio of detection at1∶100 （1%）. When CaOx being mixed with MEL-CYA complex （MEL-CYA∶ CaOx）， the two-times second derivative FTIR showed the characteristic peak on 1 740 cm-1 and 1 538 cm-1 and the minimum mass ratio of detection at 1∶500 （0.2%）. When CaOx being mixed with MEL-UA complex （MEL-UA∶ CaOx）， the two-times second derivative FTIR showed the characteristic peak at 1 117， 982 and 710 cm-1 and the minimum mass ratio of detection at 1∶250 （0.4%）. ConclusionCompare to the original spectra， the two-times second derivative FTIR can improve the detection ratio MEL in the CaOx stones from 0.2% to 1.0%. The second derivative FTIR has the unique characteristic bands and sensitive detection limit for identifying MEL in kidney stones.